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Method Article

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

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DOI:

10.3791/56147

July 27th, 2017

 ,  ,  , 

Corresponding Authors: Chih-Ying Su <c8su@ucsd.edu>

In This Article

Summary

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Abstract

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Introduction

Drosophila ORNs respond to a vast number of odorants, with widely ranging carbon chain lengths and a variety of functional groups, including esters, alcohols, ketones, lactones, aldehydes, terpenes, organic acids, amines, sulfur compounds, heterocyclics, and aromatics2,3. Odorants varied in their physicochemical features can have markedly different volatilities, indicated by the vapor pressure of the compound. Notably, biologically relevant odorants for Drosophila melanogaster differ tremendously in their volatility. For example, Ir92a ORNs respond to ammonia4, which i....

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Protocol

1. Preparation of the Hardware for at4 Recording

  1. Use a pipette puller instrument to prepare electrodes with aluminosilicate glass capillaries (O.D 1.0 mm, I.D. 0.64 mm). Blunt the tip of the reference electrode slightly with a pair of fine forceps to facilitate insertion into the clypeus of the fly (i.e. a rounded plate at the front of the fly head, above the mouthparts).
    NOTE: 7-day-old WT males (Berlin) were used in this study. Use AHL saline solution14 as the electrolyte for both electrodes.
  2. Prepare 1 L of AHL by mixing 900 mL of distilled water with 6.312 g of NaCl, 0.373 g of KCl, 0.337 g of NaHCO....

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Results

Our technique was successfully applied to determine the relative efficacy of the trans (Figure 5A) versus cis (Figure 5B) isomers of palmitoleic acid. Our representative data demonstrates that trans-palmitoleic acid is a more effective ligand for Or47b ORNs when compared to the cis isoform (Figure 5C). A single neuron was recorded from each fly, with twelve flies r.......

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Discussion

Here, we described a procedure by which the responses of Or47b ORNs to palmitoleic acid can be robustly induced and recorded. We modified a conventional long-distance odor delivery method2,7,10 to troubleshoot the problem of insufficient pheromone odorant delivery. We addressed the issue of low odorant volatility by delivering the compound via odorant cartridges, the opening of which are positioned within millimeters of the prep.......

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Ye Zhang for the help with the sample traces and Tin Ki Tsang for the help with the pictures. This work was supported by a Ray Thomas Edwards Foundation Early Career Award and an NIH grant (R01DC015519) to C.-Y.S. and NIH grants (R01DC009597 and R01DK092640) to J.W.W.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Prep Setup & Miscellaneous Materials
Pipette Puller Instrument Sutter Instruments
Novato CA USA
P97Pipette Puller
Borosilicate Glass CapillariesWorld Precision Instruments
Sarasota FL USA
1B100F-4to make holding rods
Aluminosilicate Glass Capillaries Sutter Instruments
Novato CA USA
AF100-64-10to make electrodes
Superfrost Microscope SlidesFisher Scientific
Pittsburgh PA USA
12-550-143for fly-prep station
Permanent Double Sided TapeScotch
St. Paul MN USA
NAfor fly-prep station
Upright microscopeOlympus
Shinjuku Tokyo Japan
BX51for recording rig
Plastalina modeling clayVan Aken
North Charleston SC USA
B0019QZMQQfor prep station and to stablize the holding rod
Rapid-Flow Sterile Disposable Filter Unit with SFCA Membrane, 0.45 mmNalgene
Rochester NY USA
#156-4045to sterilize AHL solution
NameCompany   Catalog NumberComments
Cartridge Materials   
200 µL pipette tip VWR
Radnor PA USA
53508-810to make odor cartridges and fly prep
Filter PaperWhatman
Maidstone Kent UK
740-Eto make odor cartridges 
Vacuum Desiccator Cole-Parmer
Vernon Hills IL USA
VX-06514-30to vaporize ethanol solvent
NameCompany   Catalog NumberComments
Odorant Materials   
cis-palmitoleic acidCayman Chemical
Ann Arbor MI USA
#10009871 (CAS # 373-49-9)Or47b odorant
trans-palmitoleic acidCayman Chemical
Ann Arbor MI USA
#9001798 (CAS # 10030-73-6)Or47b odorant
EthanolSpectrum Chemical MFG. 
New Brunswick NJ USA
E1028-500MLGLto dilute palmitoleic acid 
NameCompany   Catalog NumberComments
Rig Setup Materials   
Odorant Cartridge MicromanipulatorSiskiyou
Grants Pass OR USA
MX130Rto position the olfactometer
Flow Vision software Alicat
Tuscon AZ USA
FLOWVISIONSCsoftware to control flow rate
Mass ControllerAlicat
Tuscon AZ USA
MC-2SLPM-Dto control the flow rate for humidified air
Mass ControllerAlicat
Tuscon AZ USA
MC-500SCCM-Dto control the flow rate for odor stimulation
ClampexMolecular Devices
Sunnyvale CA USA
Ver. 10.4Data acquisition software
Air delivery tubeAce Glass
Vineland NJ USA
8802-936 to deliver humidified air
50X objective lens Olympus
Shinjuku Tokyo Japan
LMPLFL50Xrecording rig
Clampfit 10Molecular Devices
Sunnyvale CA USA
Ver. 10.4software for spike analysis 
Igor Pro 6WaveMetrics
Lake Oswego OR USA
Ver. 6.37software for data analysis 
Audio MonitorALA Scientific Instruments
Farmingdale NY USA
NPIEXB-AUDIS-08BAurally reports individual spikes
Extracellular AmplifierALA Scientific Instruments
Farmingdale NY USA
NPIEXT-02Fto increase the amplitude of electrical signals
Valve ControllerWarner Instruments   VC-8to control the opening of the valve for odor stimulation
Recording Electrode MicromanipulatorSutter Instruments
Novato CA USA
MP-285to position recording electrode
Headstage AmplifierALA Scientific Instruments
Farmingdale NY USA
EQ-16.0008to increase the amplitude of electrical signals
OscilloscopeTektronix
Beaverton OR USA
TDS2000CVisual report of individual spikes

References

  1. Lin, H. -H., et al. Hormonal modulation of pheromone detection enhances male courtship success. Neuron. 90 (6), 1272-1285 (2016).
  2. Hallem, E. A., Carlson, J. R. Coding of odors by a receptor repertoire. Cell. 125 (1), 143-160 (2006).
  3. Silbering, A. F., et al.

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Tags

Single Sensillum RecordingLow Volatility OdorantsInsect OlfactionPheromone CommunicationOdor Delivery TechniquePalmitoleic AcidOlfactory Receptor NeuronsIn Vivo Recording